The invention relates to (1) a composition of matter in which the polypeptide has the chemical structure, biophysical activity and physiologic effects of the small, hydrophobic protein SP-B with a purity of xe2x89xa795%; (2) a second composition of matter in which the polypeptide solute has the chemical structure, biophysical activity and physiologic effects of the small, hydrophobic protein SP-C with a purity of xe2x89xa795%; and (3) the methods for producing the compositions of matter.
Role of Surfactant in Pulmonary Physiology
Inhaled air containing oxygen travels through the trachea, the bronchi, and the bronchioles to the hundreds of millions of terminal alveoli. The terminal alveoli are the air spaces in the lungs where oxygen is taken up by the blood in exchange for carbon dioxide.
At the interface between the gas in the terminal alveoli and the liquid of the lung tissue, (i) oxygen diffuses into the blood from the alveoli and (ii) carbon dioxide diffuses from the blood to the alveolar air before being exhaled. To diffuse from the alveolar gas to the blood, an oxygen molecule must traverse the liquid lining the alveoli, at least one epithelial cell, the basement membrane, and at least one endothelial cell.
Pulmonary surfactant acts at the interface between alveolar gas and the liquid film lining the luminal surface of the cells of the terminal alveoli. The normal pulmonary surfactant lining is extremely thin, usually no more than 50 nm thick. Thus, the total fluid layer covering the 70 square meters of alveolar surface in an adult human is only approximately 35 ml.
For materials to be effective lung surfactants, surfactant molecules must move rapidly to the surface of the liquid. Pulmonary surfactant functions by adsorbing to the surface of the liquid covering these lining cells and changing surface tension of the alveolar fluid during the respiratory cycle.
Surface tension is a characteristic of most liquid solutions. At the interface between liquid and a gas phase, the movement of molecules at the surface of the liquid is restricted by intermolecular forces acting on those molecules. The intermolecular forces have a net direction that tends to decrease the area of the surface. The net force at the surface is referred to as surface tension. Surface tension varies with molarity, temperature and multiple solutes. Surface tension has units of force per unit length (dynes/cm or mN/m). The vector of the surface tension force is perpendicular to the plane of the interface.
The lungs of vertebrates contain surfactant, a complex mixture of lipids and protein which causes surface tensions to rise during surface expansion (inflation) and decrease during surface compression (deflation). During lung deflation, surfactant decreases surface tension to xe2x89xa61 mN/m, so that there are no surface forces that would otherwise promote alveolar collapse. Aerated alveoli that have not collapsed during expiration permit continuous O2 and CO2 transport between blood and alveolar gas and require much less force to inflate during the subsequent inspiration.
In order to attain sufficient uptake of oxygen by the blood and excretion of carbon dioxide from the blood, an animal""s lungs must ventilate the terminal alveoli simultaneously and evenly. Either unsynchronized or uneven ventilation will prevent sufficient oxygen uptake into the circulating blood and result in the retention of carbon dioxide in the body.
During inflation, lung surfactant increases surface tension as the alveolar surface area increases. A rising surface tension in expanding alveoli opposes over-inflation in those airspaces and tends to divert inspired air to less well-aerated alveoli, thereby facilitating even lung aeration.
Surfactant Deficiency or Dysfunction
Although the exact composition and physical characteristics of natural lung surfactant have not been determined, material isolated from the lumen of lungs, termed natural surfactant, contains a mixture of phospholipids, neutral lipids, and proteins. (Jobe A, Ikegami M, Surfactant for the treatment of respiratory distress syndrome. Am Rev Respir Dis, 1987; 136:1256-75.) The phospholipids are not specific to surfactant, but are also present in other biologic materials, particularly membranes. The predominant phospholipids in surfactant, however, are disaturated phosphatidylcholines which are present in low concentrations in most membranes. Among the proteins found in the lung lumen are mucoproteins, plasma proteins, and lung specific proteins. These lung specific proteins are described in more detail in Section 2.3.
The alveoli are lined with epithelial cells that have a role in producing surfactant, maintaining the activity of surfactant, and preventing the inactivation of surfactant. The epithelial cells form a continuous, tight barrier that normally prevents entry into the alveoli of molecules from the circulation that can inhibit surfactant.
The alveolar epithelium consists of at least two types of alveolar cells, referred to as type I and type II alveolar cells. The type II alveolar cells normally synthesize both the phospholipids and proteins that are in lung surfactant, store newly synthesized material in the intracellular inclusion bodies, secrete the surfactant into the alveolar space, absorb surfactant from the alveolar space, and metabolize material re-incorporated into the type II cell. The role of type I cells in surfactant function has not yet been identified.
Lung surfactant is normally synthesized at a very low rate until the last six weeks of fetal life. Human infants born more than six weeks before the normal term of a pregnancy have a high risk of being born with inadequate amounts of lung surfactant and inadequate rates of surfactant synthesis. The more prematurely an infant is born, the more severe the surfactant deficiency is likely to be. Severe surfactant deficiency can lead to respiratory failure within a few minutes or hours of birth. The surfactant deficiency produces progressive collapse of alveoli (atelectasis) because of the decreasing ability of the lung to expand despite maximum inspiratory effort. As a result, inadequate amounts of oxygen reach the infant""s blood.
Endogenous surfactant production typically accelerates after birth, even in quite premature infants. If the infant survives the first few days, lung surfactant status generally becomes adequate.
Additional causes of respiratory failure from surfactant dysfunction have been reported due to defects in surfactant synthesis (congenital protein B deficiency), or in secretion or metabolism of surfactant (alveolar proteinosis). In addition, lung surfactant can be inhibited and inactivated in vitro by a variety of proteins, cell wall phospholipids, enzymes, and other products of inflammatory responses.
Injury to juvenile and adult animals can also inactivate surfactant and produce a respiratory failure with a similar pathophysiology to the surfactant deficiency in premature infants. This respiratory failure is often referred to as the Adult (or Acute) Respiratory Distress Syndrome, ARDS. This syndrome results from several simultaneous pathologic processes, one of which is generalized inhibition of the extra-cellular surfactant in the alveolar space plus dysfunction of the type II alveolar cells which adversely affect the synthesis, secretion, or metabolism of surfactant.
Current treatment of respiratory failure includes supplementation of oxygen, mechanical ventilation, and instillation or aerosolization of materials with lung surfactant activity. Some patients die from respiratory failure despite current treatments, some survive with permanently damaged lungs, and other patients recover after prolonged therapy.
Hydrophobic Surfactant Proteins
Lung surfactants are complex materials composed of multiple molecules that interact physically, without combining chemically, to achieve their biologic activity. Natural lung surfactant contains lipids and proteins. There are two types of lung surfactant proteins, hydrophilic and hydrophobic. The two hydrophilic surfactant proteins identified to date, named SP-A and SP-D, are water soluble, chloroform insoluble, glycosolated, have polypeptide chains  greater than 25,000 Daltons and are not essential for lung surfactant activity at the air: gas interface. The hydrophobic proteins, named SP-B and SP-C are not water soluble, are chloroform soluble, are not glycosolated, have polypeptide chains less than 26,000 Daltons after post translational modification to their active form, and are essential for normal biophysical and biologic activity of lung surfactant.
All of these surfactant proteins have been sequenced. The SP-C sequence has been determined in mice, dogs, rats, humans and cows and rats. The homology between the sequence of protein SP-C in humans and the sequence of protein SP-C in other species is  greater than 80%. The SP-B protein sequence has been determined for humans, dogs, rats, rabbits, mice and cows. Most of the sequence identity of SP-B is shared throughout species. (Whitsett J A and Baatz J E. Hydrophobic surfactant proteins SP-B and SP-C: molecular biology, structure and function in Robertson B, Van Golde L M G, Batenburg J J eds. Pulmonary Surfactant: From Molecular Biology to Clinical Practice Elsivier, New York, 1992, pp. 55-75.) Both SP-B and SP-C are synthesized initially as proproteins which comprise the active polypeptide chain plus additional amino acids added to one or both ends. After synthesis, the proproteins of SP-B and SP-C are modified by proteolytic processes to remove the additional amino acids, thereby yielding the active molecule. This process is referred to as post-translational modification.
Several forms of both the SP-B and SP-C active proteins are observed naturally. Monomers, single molecules, are observed as are oligomers or small numbers of chains bound together. Some oligomers are formed by sulfide bridges (ones that are broken into monomers by reducing agents) and some bound together by other, non-sulfide, bonds. Oligomers that are formed by sulfide bridges can be separated into monomers by reducing agents. It is unknown whether the proteins as synthesized or any intermediates have biologic activity. It is also unknown whether differences in activity exist between different oligomers, or between monomers and oligomers of these proteins.
Hydrophobic Proteins in Lung Surfactant Drugs
Eight different surfactants have been developed to treat newborn infants with Respiratory Distress Syndrome, RDS. Two lung surfactant drugs have lipids, but no surfactant proteins (Exosurf, ALEC). Three surfactant drugs have lipids and significant amounts of SP-C but low levels of SP-B (Survanta, Surfacten, Curosurf). Three others have significant amounts of both hydrophobic surfactant proteins (Infasurf, Alveofact, bLES). Infasurf and Alveofact are more biophysically active than Survanta or Curosurf, which in turn are more biophysically active than Exosurf. (Hall S B, Venkitaram A R, Whitsett J A, et al.: Importance of hydrophobic apoproteins as constituents of clinical exogenous surfactants. Am Rev Respir Dis 1992; 145:24-30; Seeger W., Grube C. Gunther A. Schmidt R. Surfactant inhibition by plasma proteins: differential sensitivity of various surfactant preparations, Eur Respir J 1993: 6:971-977.) Infasurf is more biologically active than Survanta, and Survanta is more biologically active than Exosurf, measured as either biophysical or physiologic activity. (Cummings J J, Holm B A, Hudak M L, Hudak B B, Ferguson W H, Egan E A: Controlled clinical comparison of four different surfactant preparations in surfactant-deficient preterm lambs. Am Rev Respir Dis 1991; 145:999-1003. Mizuno K, Ikegami M, Chen C M, Ueda T, Jobe A H. Surfactant protein-B supplementation improves in vivo function of a modified natural surfactant. Pediatr Res 1995; 37:271-276.) Clinically, Infasurf is more effective than Survanta or Exosurf, and Survanta is more effective than Exosurf. (Hudak M L, Farrell E E, Rosenberg A A et al. A multicenter randomized masked comparison trial of natural versus synthetic surfactant for the treatment of respiratory distress syndrome. J Pediatr 1996; 128:396-406; Bloom B T, Kattwinkel J, Hall R T et al. Comparison of Infasurf (calf lung surfactant extract) to Survanta (beractant) in the treatment and prevention of RDS. Pediatrics in Press, July 1997; Hudak M L, Cummings J J, Jung A L et al. Infasurf v. Exosurf for the prevention of neonatal respiratory distress syndrome: a multicenter randomized masked comparison trial. Pediatrics in press, July 1997.) The differences in activity of lung surfactants are associated with the amount and type of hydrophobic proteins they contain.
Preparation of Purified Hydrophobic Proteins
Surfactant proteins have been separated from surfactant lipids and SP-B has been separated from SP-C for academic investigations into protein metabolism and function. (Kogishi F, Kurozumi Y Fukite et al. Isolation and partial characterization of human low molecular weight protein associated with pulmonary surfactant. Am Rev Respir Dis 1988; 137:1426-1431; Mathialgian N, Possmayer F. Low molecular-weight hydrophobic proteins from pulmonary surfactant. Biochem Biophys Acta 1990: 1045:121-127; Takahshi A, Waring A J, Amirkhanian J, et al. Structure function relationships of bovine pulmonary surfactant proteins: SP-B and SP-C. Biochim Biophys Acta 1990, 1044:43-49; Wang Z, Gurel G, Baatz J E, Notter R H. Lung surfactant proteins SP-B and SP-C are not synergistic in surface active interactions with phospholipids J. Lipid Res. 1996; 37:1749-1760.) These methods describe organic extraction of natural surfactant followed by single or multiple column chromatography processes to separate hydrophobic proteins from lipids. Separation of the two hydrophobic surfactant proteins from each other has utilized repeated, additional column chromatography and/or preparative SDS PAGE, and/or reverse phase HPLC. Evaluation of the purity of the resulting proteins has been qualitative. Protein solutions produced by these methods have not reported yields. A process using only differential solubility in organic solvents has been described, but the method results in detectable levels of more than one polypeptide chain in the xe2x80x9cpurexe2x80x9d SP-B. (Beers M F, Bates S R, Fisher A B. Differential extraction for the rapid purification of bovine surfactant protein B. Am J Physiol 1992; 262:L773-L778.) None of these methods presents a practical method for securing significant quantities (milligrams or grams per procedure) of hydrophobic surfactant proteins, i.e., (1) a xe2x89xa750% yield of SP-B and/or SP-C from an amount of SP-B and/or SP-C in a reference sample or (2) SP-B and/or SP-C of xe2x89xa795% purity from biologically generated sources.
Definitions of Abbreviations
As used in this description, the following abbreviations have the following meanings:
ARDSxe2x80x94Adult (or Acute) Respiratory Distress Syndrome
xc2x0 C.xe2x80x94degrees celsius
cmxe2x80x94centimeter
Dxe2x80x94Dalton
DPPCxe2x80x94dipalmatoylphosphatidylcholine
gxe2x80x94gravity
 greater than xe2x80x94greater than
H2Oxe2x80x94molecular abbreviation for water
HPLCxe2x80x94high pressure liquid chromatography
kDxe2x80x94kiloDalton
kgxe2x80x94kilogram
 less than xe2x80x94less than
1xe2x80x94liter
LPCxe2x80x94lysophosphatidylcholine
mxe2x80x94meter
xcexclxe2x80x94microliter
xcexcmxe2x80x94micrometer (micron)
mgxe2x80x94milligram
minxe2x80x94minute
mlxe2x80x94milliliter
mmxe2x80x94millimeter
mMxe2x80x94milliMolar
mNxe2x80x94milliNewton
Ngxe2x80x94nanogram
Nxe2x80x94Newton
O2xe2x80x94molecular abbreviation for oxygen
PBSxe2x80x94pulsating bubble
PCxe2x80x94phosphatydilcholine
PC/DSxe2x80x94phosphatidylcholinexe2x80x94disaturated
PExe2x80x94phosphatydilethanolamine
PGxe2x80x94phosophatidylglycerol
pHxe2x80x94negative logarithm of hydrogen ion concentration
PIxe2x80x94phosphatidylinositol
PLxe2x80x94phospholipid
RDSxe2x80x94Respiratory Distress Syndrome
rpmxe2x80x94revolutions per minute
SDS PAGExe2x80x94sodium dodecylsulfate polyacrylimide gel electrophoresis
SP-Axe2x80x94surfactant protein A
SP-Bxe2x80x94surfactant protein B
SP-Cxe2x80x94surfactant protein C
SP-Dxe2x80x94surfactant protein D
SPHxe2x80x94sphingomyelin
SN-1xe2x80x94position 1 of glycerol molecule
SN-2xe2x80x94position 2 of glycerol molecule
volxe2x80x94volume
New Compositions of Matter and Methods for Producing the Same
The invention is a new composition of matter which is a purified preparation of each of the SP-C and SP-B lung surfactant hydrophobic proteins which retain their full biophysical and physiologic activity. Each solution has xe2x89xa795% of peptides that are specific to a single protein by N-terminal amino acid sequencing, total amino acid analysis and SDS polyacrylamide gel electrophoresis. The new materials can be used to fabricate novel pharmacologic agents with specific SP-B and/or SP-C contents relative to other components.
According to another object of the present invention there is provided a method for separating hydrophobic proteins SP-B and SP-C from aqueous sources containing other biologic molecules.
According to another object of the present invention there is provided a method for separating hydrophobic proteins SP-B and SP-C from organic solvent solutions containing other biologic molecules.
According to another object of the present invention, there is provided a high-yield method for separating SP-B and SP-C from biological substances.
Other objects and advantages of the present invention will be apparent to those skilled in the art.